Large stress/heat shock proteins (LSPs) that are complexed in vitro with clinically relevant tumor protein antigens by heat shock have demonstrated highly potent antitumor efficacy in animal models. However, the molecular basis of immunoregulatory features of LSPs remains largely undetermined. Our initial work led to an unexpected finding that enhanced cross-presentation of melanoma protein antigen (Ag) by exogenously delivered LSP involved the endoplasmic reticulum-associated degradation (ERAD) machinery, an intracellular protein quality control system. In addition, for the first time we have discovered that LSPs actively interact with pathogen-derived molecules or PAMPs, resulting in synergistic activation of intracellular NOD-like receptor (NLR)/inflammasome. These observations, which implicate the molecular chaperoning underlying the essential immunologic activities of LSPs, lead us to hypothesize that LSPs are capable of engaging and bridging both innate and adaptive compartments through their highly efficient chaperoning functions. The overall objective of this application is to investigate the highly novel aspects of LSPs in interacting with a self tumor protein Ag and PAMP molecules, as well as distinct immune consequences. Furthermore, LSP-primed cellular responses under physiologically relevant fever-like thermal stress conditions will be assessed. As a consequence of these studies, a novel approach to further enhance the therapeutic potency of the chaperone vaccine regimen by integrating both Ag target and PAMP molecules will be evaluated. The following specific aims will be pursued to achieve the project goal: 1) Determine the protein quality control mechanisms underlying LSP-enhanced cross-presentation of tumor protein Ag. 2) Determine the capability of LSP to interact with PAMPs and modify the resultant immune outcomes. 3) Determine the antitumor efficacy of a modified chaperone vaccine regimen incorporating a PAMP-derived 'danger' signal. Given the common chaperoning property shared by different classes of heat shock proteins, successful completion of these studies will provide important insights into the unique biological activities of these ancient molecules. The proposed studies will also shed new light on the role of mild thermal stress/hyperthermia in the host response, which could be exploited therapeutically for immune modulation. A detailed understanding of the LSP action in host defense mechanisms should provide more effective and safe strategies for targeting cancer and other diseases of clinical importance.